Podocytes are specialized cells of the kidney involved in filtering waste products from the blood. However, in various nephrotic syndromes, these cells become compromised and their foot processes detach from the underlying glomerular basement membrane (GBM). This detachment is thought to be the major cause of disease symptoms such as proteinuria and will eventually lead to kidney failure in many cases. Understanding how the podocytes form and retain their foot process attachments to the GBM is therefore an important area of research. The experiments in this proposal are designed to characterize molecules involved in maintaining these attachments, and may lead to novel therapeutic targets for various kidney diseases. These goals directly relate to the mission of the NIH/NIDDK to improve human health through novel research, in this case specifically related to renal function and disease. Podocyte-GBM attachment factor (PGAF) is a novel protein required for the attachment of podocyte foot processes to the GBM. It is a downstream target of Wilms' tumor suppressor-1 (WT1) and binds capnsi, the small subunit of the cysteine protease calpain. As active calpains promote detachment of cellular adhesions, the current hypothesis is that PGAF inhibits calpain activity in podocytes to prevent foot process detachment. The aims of this proposal are to use a combination of in vitro and in vivo techniques to investigate the role of PGAF and calpains in the maintenance of podocyte attachments to the GBM. In vivo studies will be conducted on a combination of zebrafish and mouse models. As the embryonic zebrafish kidney provides a simple, rapid and easily manipulatable system in which to study podocyte function, our primary in vivo analysis of PGAF and calpain function will be carried out in this system. Techniques including morpholino-based gene knockdown and mRNA over-expression will be used to perturb gene function in embryos. Electron microscopy analysis of podocytes and assessment of glomerular filtration ability will be used to analyze the resulting mutant embryos. In addition, PGAF loss-of-function mice will be generated and assessed using similar techniques. In vitro analysis will involve biochemical characterization of protein interactions and knockdown/overexpression of PGAF in cultured murine podocyte cells. Relevance to public health: The kidney filters waste from the blood by using specialized cells called podocytes. However, these cells can become damaged by conditions such as diabetes or various kidney diseases. Our research will examine how podocytes form and are maintained, which will facilitate our understanding of what happens to them during disease, and potentially lead to new therapies.